Uppsala BioCenter, Department of Molecular Science, Swedish University of Agricultural Sciences, Uppsala, Sweden.
Faculty of Chemistry and Chemical Technology, Department of Chemistry and Biochemistry, University of Ljubljana, Ljubljana, Slovenia.
Methods Mol Biol. 2022;2447:13-20. doi: 10.1007/978-1-0716-2079-3_2.
Type II metacaspases (MCAs) are proteases, belonging to the C14B MEROPS family. Like the MCAs of type I and type III, they preferentially cleave their substrates after the positively charged amino acid residues (Arg or Lys) at the P1 position. Type II MCAs from various higher plants have already been successfully overexpressed in E. coli mostly as His-tagged proteins and were shown to be proteolytically active after the purification. Here we present a protocol for expression and purification of the only type II MCA from the model green alga Chlamydomonas reinhardtii. The two-step purification, which consists of immobilized metal affinity chromatography using cobalt as ion followed by size-exclusion chromatography, can be performed in 1 day and yields 4 mg CrMCA-II protein per liter of overexpression culture.
II 型 metacaspases (MCAs) 是一种蛋白酶，属于 C14B MEROPS 家族。与 I 型和 III 型的 MCAs 一样，它们优先在 P1 位置的正电荷氨基酸残基（Arg 或 Lys）之后切割其底物。来自各种高等植物的 II 型 MCAs 已成功在大肠杆菌中过表达，主要作为 His 标记的蛋白质表达，在纯化后显示出蛋白水解活性。在这里，我们提供了从模式绿藻莱茵衣藻中唯一的 II 型 MCA 进行表达和纯化的方案。两步纯化过程包括使用钴作为离子的固定化金属亲和层析，然后是大小排阻层析，可在 1 天内完成，每升过表达培养物可产生 4 毫克 CrMCA-II 蛋白。
